Journal: Cellular and Molecular Life Sciences

Article Title: CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

doi: 10.1007/s00018-022-04201-9

Figure Lengend Snippet: Cellular localization and axonal transport of endogenous IGF-1 in DRG. A , B DRG tissue sections or C cultured DRG neurons from control rats underwent RNA FISH assay for IGF-1 mRNA detection and localization. A punctate pattern of IGF-1 mRNA is evident in neurons and a limited number of associated glia. As a control, tissue sections and cells were exposed to RNase enzyme before hybridizing with IGF-1 probes. Images are magnified for clarification. As a control, tissue sections and cells were exposed to RNase enzyme before hybridizing with IGF-1 probes. IGF-1 mRNA punctate labeling is shown in A neurons and B glia in DRG tissue sections. In C , cultured DRG neurons and associated glia showed punctate staining for IGF-1. Boxes 1–3 are magnified parts of the figure shown for clarity. Arrows show glial and neuronal IGF-1 in box1, neuronal IGF-1 in box2 and glial IGF-1 in box3. In D , publicly available scRNA-Seq normalized data (RPM) from 240 to 480 cells was analyzed and relative transcript levels of Igf1 and Igf1r were plotted for each DRG subpopulation: NF cluster (myelinated neurons), PEP cluster (peptidergic nociceptors), NP cluster (non-peptidergic nociceptors), TH cluster (type-C low-threshold mechanoreceptors), SC cluster (Schwann cells) and SGC cluster (satellite glial cells). In E , a 12-h double ligature (1 cm width) experiment was carried out on rat sciatic nerve with four regions proximal 1 (P1), P2, distal 1 (D1) and D2 analyzed for IGF-1 levels. Data are mean ± SEM of N = 3–4. * p < 0.05 or ** p < 0.01; analyzed by one-way ANOVA with Tukey’s post hoc test

Article Snippet: All LNP–siRNA, LNP–GFP (AG13105-CH, Sino Biological Inc., Beijing, China), LNP–IGF-1 (HG29626-NH, Sino Biological Inc., Beijing, China) and LNP–CEBPβ (Addgene, USA; plasmid # 12557) overexpressing plasmids were formulated using Neuro9 (Precision NanoSystems, Vancouver, BC, Canada) transfection kit following microfluidics mixing method using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Vancouver, BC, Canada) following supplied protocol.

Techniques: Cell Culture, Clarification Assay, Labeling, Staining

Journal: Cellular and Molecular Life Sciences

Article Title: CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

doi: 10.1007/s00018-022-04201-9

Figure Lengend Snippet: The level of endogenous IGF-1 was reduced in DRG tissue from type 1 and type 2 diabetic rodents, in part, via hyperglycemia-activated polyol pathway activity and restored by exogenous hIGF-1. In A – D DRG tissues from control (Ctrl), hIGF-1-treated (STZ-Db + hIGF-1), untreated STZ-diabetic (STZ-Db), Zucker diabetic fatty (ZDF) rats and db/db mice were homogenized and underwent A qRT-PCR or B – D ELISA for IGF-1 detection. In E , DRG neurons derived from Ctrl and STZ-Db rats were cultured and media was collected after 2 days to measure secreted IGF-1 protein. DRG neurons derived from control ( F , G , H ) or diabetic ( I ) rats were cultured in the presence of 25 mM glucose with/without insulin or hIGF-1 treatment or in the presence of 5 mM glucose. RNA was extracted and utilized for real-time PCR assay. In G , 25 mM mannitol was used to control for osmotic pressure compared with 25 mM D-glucose. In H , sorbinil (10 and 100 µM), an aldose reductase inhibitor (ARI), was applied. Data are mean ± SEM of N = 3–6; * p < 0.05 or ** p < 0.01 or **** p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s or Tukey’s post hoc test

Article Snippet: All LNP–siRNA, LNP–GFP (AG13105-CH, Sino Biological Inc., Beijing, China), LNP–IGF-1 (HG29626-NH, Sino Biological Inc., Beijing, China) and LNP–CEBPβ (Addgene, USA; plasmid # 12557) overexpressing plasmids were formulated using Neuro9 (Precision NanoSystems, Vancouver, BC, Canada) transfection kit following microfluidics mixing method using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Vancouver, BC, Canada) following supplied protocol.

Techniques: Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences

Article Title: CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

doi: 10.1007/s00018-022-04201-9

Figure Lengend Snippet: IGF-1 neutralizing antibody or IGF-1 targeting siRNAs reduced IGF-1Rβ and Akt phosphorylation and diminish neurite outgrowth. DRG neurons from control rats were cultured, treated with different doses of IGF-1 neutralizing antibody and underwent A , B Western blotting for Akt phosphorylation and IGF-1Rβ or C , D neurite outgrowth measurement. In A , B , total protein bands were used for normalization. In C , D , normal goat IgG was used as a control antibody. In E – G , DRG neurons from control rats were cultured, treated with different doses of two IGF-1 targeting siRNAs and underwent E real-time PCR assay for IGF-1 or F , G neurite outgrowth measurement. In E , four transcript variants (a pair of primers designed for the detection of two variants 1 and 2 or 3 and 4) of IGF-1 that produce protein were analyzed. In F , G , exogenous hIGF-1 was added alone or along with IGF-1 knock-down as control groups. Data are mean ± SEM of N = 4; * p < 0.05 or ** p < 0.01 or **** p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s post hoc test

Article Snippet: All LNP–siRNA, LNP–GFP (AG13105-CH, Sino Biological Inc., Beijing, China), LNP–IGF-1 (HG29626-NH, Sino Biological Inc., Beijing, China) and LNP–CEBPβ (Addgene, USA; plasmid # 12557) overexpressing plasmids were formulated using Neuro9 (Precision NanoSystems, Vancouver, BC, Canada) transfection kit following microfluidics mixing method using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Vancouver, BC, Canada) following supplied protocol.

Techniques: Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences

Article Title: CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

doi: 10.1007/s00018-022-04201-9

Figure Lengend Snippet: IGF-1-overexpressing plasmid enhanced glycolysis, mitochondrial respiration, and neurite outgrowth. DRG neurons from control rats were cultured, transfected with different doses of hIGF-1 (transcript variant 4)-overexpressing plasmid and 200 ng control GFP plasmid (ctrl). In A , B , mitochondrial OCR was measured in live neurons after 6 h. In C – F , glycolysis parameters and total neurite outgrowth were calculated. Total protein in mg was used to normalize OCR and ECAR data. Data are mean ± SEM of N = 4–6; * p < 0.05 or ** p < 0.01 or *** p < 0.001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s post hoc test

Article Snippet: All LNP–siRNA, LNP–GFP (AG13105-CH, Sino Biological Inc., Beijing, China), LNP–IGF-1 (HG29626-NH, Sino Biological Inc., Beijing, China) and LNP–CEBPβ (Addgene, USA; plasmid # 12557) overexpressing plasmids were formulated using Neuro9 (Precision NanoSystems, Vancouver, BC, Canada) transfection kit following microfluidics mixing method using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Vancouver, BC, Canada) following supplied protocol.

Techniques: Plasmid Preparation, Cell Culture, Transfection, Variant Assay

Journal: Cellular and Molecular Life Sciences

Article Title: CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

doi: 10.1007/s00018-022-04201-9

Figure Lengend Snippet: NFAT1 and CEBPβ transcription factors activated IGF-1 gene expression and were less enriched at the Igf1 promoter in DRG tissue derived from diabetic rats. About A 1.2 kb of IGF-1 gene promoter region was chosen to design ChIP assay. Five regions were used for amplification and each pair of primers was designed to include one transcription factor-binding site. In B , DRG tissues derived from adult control and STZ-diabetic rats underwent ChIP assay using NFAT1 and CEBPβ antibodies for pull down followed by IGF-1 promoter region (five regions in total stats for three are shown) amplification using ChIP-qRT-PCR analysis. In C , diagram of the mutated transcription factor (NFAT1 and CEBPβ) binding sites on promoter region of Igf1 gene is given. A total of 4 binding sites (3a for NFAT1-binding site 3, 3b for CEBPβ-binding site 3, 5 for NFAT1- and CEBPβ-binding site 5) from ( A ) were mutated. In D , luciferase activity of the mutated and wild type IGF-1 promoter was measured in HEK293 cells. In E , DRG neurons from STZ-diabetic (Db) rats were transfected with NFAT1, CEBPβ or both, and different transcript variants (Tv1,2,3,4) of IGF-1 were measured using qRT-PCR. Data are mean ± SEM of N = 4–5 animals or N = 4–6 culture groups; * p < 0.05 or ** p < 0.01 or **** p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Tukey’s post hoc test

Article Snippet: All LNP–siRNA, LNP–GFP (AG13105-CH, Sino Biological Inc., Beijing, China), LNP–IGF-1 (HG29626-NH, Sino Biological Inc., Beijing, China) and LNP–CEBPβ (Addgene, USA; plasmid # 12557) overexpressing plasmids were formulated using Neuro9 (Precision NanoSystems, Vancouver, BC, Canada) transfection kit following microfluidics mixing method using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Vancouver, BC, Canada) following supplied protocol.

Techniques: Expressing, Derivative Assay, Amplification, Binding Assay, Quantitative RT-PCR, Luciferase, Activity Assay, Transfection

Journal: Cellular and Molecular Life Sciences

Article Title: CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

doi: 10.1007/s00018-022-04201-9

Figure Lengend Snippet: IGF-1 knock-down suppressed CEBPβ upregulation of mitochondrial function and neurite outgrowth. DRG neurons from STZ-diabetic (Db) rats were cultured, transfected with pCEBPβ–LNP, pGFP–LNP or siIGF1–LNP, and underwent A , B mitochondrial respiration assay or C , D neurite outgrowth measurement. In A , B , total protein in mg was used to normalize OCR data. In E , ELISA assay was used to measure IGF-1 levels in the same condition as in C . Data are mean ± SEM of N = 3–5; * p < 0.05 or ** p < 0.01 or *** p < 0.001; analyzed by one-way ANOVA with Tukey’s post hoc test

Article Snippet: All LNP–siRNA, LNP–GFP (AG13105-CH, Sino Biological Inc., Beijing, China), LNP–IGF-1 (HG29626-NH, Sino Biological Inc., Beijing, China) and LNP–CEBPβ (Addgene, USA; plasmid # 12557) overexpressing plasmids were formulated using Neuro9 (Precision NanoSystems, Vancouver, BC, Canada) transfection kit following microfluidics mixing method using the NanoAssemblr Benchtop instrument (Precision NanoSystems, Vancouver, BC, Canada) following supplied protocol.

Techniques: Cell Culture, Transfection, Respiration Assay, Enzyme-linked Immunosorbent Assay